Nucleic acid structure having introduced hydrophobic moiety, and preparation method therefor

Abstract

The nucleic acid structure according to the present invention can increase capture efficiency in a drug delivery system by allowing a hydrophobic moiety to be introduced through hybridization of a desired nucleic acid molecule with another nucleic acid molecule that contains the hydrophobic moiety and a sequence complementary to an arbitrary sequence present in the desired nucleic acid molecule, thereby enabling in vivo delivery efficiency, stability and expression efficiency to be improved, and thus can be effectively used as an improved drug delivery system and biopharmaceutical.

Description

Nucleic acid structure introducing hydrophobic residues and method for producing the same

[0001] The present invention relates to a nucleic acid structure having a hydrophobic residue introduced therein and a method for producing the same, and more particularly, to a nucleic acid structure having improved in vivo delivery efficiency, stability, and expression efficiency by introducing a hydrophobic residue.

[0002] Many diseases are caused by increased expression of disease genes or abnormal activity due to mutations, which are caused by various factors. Messenger RNA (mRNA) is a substance that contains genetic information before protein synthesis. mRNA can be accessed by various therapeutics, making it useful as a preventive or therapeutic vaccine. It can also synthesize missing proteins. The advantages of mRNA therapeutics are that, compared to DNA, they do not require delivery to the nucleus. They are also safer because they are not integrated into the genome, preventing permanent genetic diseases. Furthermore, mRNA can synthesize missing proteins within cells, inaccessible to protein therapeutics. mRNA can vary in size depending on the protein it expresses, and exists as a single strand. Created from DNA, mRNA escapes from the nucleus into the cytoplasm, where it interacts with ribosomes to produce proteins.

[0003] While mRNA is attracting attention as a next-generation gene therapy, it is vulnerable to degradation by nucleases in the bloodstream and has limited access to intracellular compartments where translation machinery resides. Attempts are being made to introduce lipid nanoparticles formed from cationic lipids, along with other lipid components such as neutral lipids, cholesterol, PEG, PEGylated lipids, and oligonucleotides, to block mRNA degradation in the bloodstream and promote cellular uptake of nucleic acids.

[0004] When using lipid-based delivery vehicles such as liposomes or lipid nanoparticles, mRNA is typically either adsorbed to the outside or encapsulated inside. In particular, when mRNA is adsorbed to the outside of the delivery vehicle, it is generally known to exist as an aggregate of liposomes and nucleic acid, rather than a single liposome. Depending on the combination of lipids, the state of the nucleic acid, and the ratio of nucleic acid to liposome, the adsorption capacity varies and stability is affected. Furthermore, because a large amount of lipid is transferred into the body for nucleic acid delivery, which can be accompanied by various side effects, there is a need to reduce the amount of lipid relative to mRNA.

[0005] Accordingly, the inventors of the present invention have completed the present invention by introducing hydrophobic residues into a nucleic acid structure to develop a formulation with a reduced amount of lipid compared to mRNA, thereby increasing the capture efficiency within a drug delivery system, and thereby confirming an improvement in in vivo delivery efficiency, stability, and expression efficiency.

[0006] An object of the present invention is to provide a nucleic acid structure.

[0007] Another object of the present invention is to provide a composition comprising the nucleic acid structure and lipid nanoparticles.

[0008] Another object of the present invention is to provide a vaccine composition comprising the nucleic acid structure.

[0009] Another object of the present invention is to provide a method for producing the nucleic acid structure.

[0010] To achieve the above object, the present invention provides a nucleic acid structure comprising (a) an mRNA comprising a coding region for mRNA expression of a target gene, a poly(A) tail, and an additional sequence at the end of the poly(A) tail; and (b) a DNA oligo complementarily binding to a portion of the poly(A) tail sequence and an additional sequence at the end of the poly(A) tail, and having a hydrophobic residue introduced therein.

[0011] The present invention also provides a composition comprising the nucleic acid structure and lipid nanoparticles.

[0012] Additionally, the present invention provides a vaccine composition comprising the nucleic acid structure.

[0013] In addition, the present invention provides a method for producing a nucleic acid structure, comprising: 1) a step of synthesizing mRNA by performing in vitro transcription using a vector containing mRNA including a coding region for mRNA expression of a target gene, a poly(A) tail, and an additional sequence at the end of the poly(A) tail as a template; 2) a step of producing a DNA oligo complementarily binding to a portion of the poly(A) tail sequence and an additional sequence at the end of the poly(A) tail and introducing a hydrophobic residue; and 3) a step of annealing the mRNA of step 1) and the DNA oligo of step 2).

[0014] The present invention relates to a nucleic acid structure having a hydrophobic residue introduced therein and a method for producing the same. The nucleic acid structure according to the present invention can increase the capture efficiency in a drug delivery system by introducing a hydrophobic residue by hybridizing another nucleic acid molecule having a hydrophobic residue and a complementary sequence to any sequence present in a desired nucleic acid molecule, thereby improving the in vivo delivery efficiency, stability, and expression efficiency, and thus can be usefully used as an improved drug delivery system and biopharmaceutical.

[0015] Figure 1 shows the results of electrophoresis using a 1% agarose gel after performing PCR using a DNA vector (Luciferase-pcDNA3) containing a T7 promoter, 5' UTR, an open reading frame (ORF) encoding the Luciferase protein, and 3' UTR as a template.

[0016] Figure 2 shows the results of electrophoresis using a 15% PAGE gel of mRNA before annealing and two types of Chol-DNA Oligo (5'Chol-DNA Oligo and 5',3'Chol-DNA Oligo) after annealing.

[0017] Figure 2b is a diagram showing an RNA / DNA hybrid structure in which two Chol-DNA Oligos complementary to the 3' end of mRNA are each annealed.

[0018] Figure 3 is a diagram showing the results of electrophoresis after performing an alkylation process to produce alkyl PS (phosphorothioate)-DNA Oligo.

[0019] Figure 4 is a diagram showing the alkylation mechanism of PS-DNA Oligo.

[0020] Figure 5 shows the results of electrophoresis using a 15% PAGE gel of three mRNAs after annealing and three PS-DNA Oligos (PS-65 DNA Oligo, PS-65A DNA Oligo, PS-65B DNA Oligo) before annealing.

[0021] Figure 6 shows the total lipid content of Moderna®'s mRNA vaccine formulation (L1) and formulations with reduced amounts of lipids relative to mRNA in Moderna® formulations (L2, L3, L4) (blue: lipid ratio, red: NP ratio).

[0022] Figure 7a is a diagram showing the expected particle-intake form when mRNA (mRNA) is encapsulated into lipid nanoparticles without introducing hydrophobicity into the mRNA.

[0023] Figure 7b is a diagram showing the expected particle-encapsulation form when mRNA with cholesterol introduced (mRNA / Chol) is encapsulated in lipid nanoparticles.

[0024] Figure 7c is a diagram showing the expected particle-encapsulation form when mRNA with an alkyl group introduced (mRNA / alkyl PS) is encapsulated into lipid nanoparticles.

[0025] Figure 8 is a graph showing the results for the encapsulation rate, diameter, and PDI of each lipid nanoparticle encapsulating single-stranded mRNA and mRNA having an RNA / DNA-Chol hybrid structure at the 3' end of the mRNA.

[0026] Figure 9 is a graph showing the results for the encapsulation rate, diameter, and PDI of each lipid nanoparticle encapsulating single-stranded mRNA and three types of mRNA having an RNA / alkyl PS-DNA hybrid structure at the 3' end of the mRNA.

[0027] Figure 10a shows the results of confirming the expression of Firefly Luciferase protein after encapsulating single-stranded mRNA and mRNA having an RNA / DNA-Chol hybrid structure in which a DNA oligo with cholesterol attached to the 3' end of the mRNA was introduced into lipid nanoparticles in HEK-293T cells.

[0028] Figure 10b shows the results of confirming the expression of Firefly Luciferase protein after encapsulating single-stranded mRNA and mRNA having an RNA / DNA-Chol hybrid structure in which a DNA oligo with cholesterol bound to the 3' end of the mRNA was introduced into lipid nanoparticles in HeLa cells.

[0029] Figure 11 shows the results of confirming the expression of Firefly Luciferase protein after encapsulating single-stranded mRNA and mRNA having an RNA / alkyl PS-DNA hybrid structure with an alkyl group introduced at the 3' end of the mRNA into lipid nanoparticles in HepG2 cells.

[0030] Hereinafter, the present invention will be described in detail.

[0031] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. In general, the nomenclature used herein is well known and commonly used in the art.

[0032] The present invention provides a nucleic acid construct comprising (a) an mRNA comprising a coding region for mRNA expression of a target gene, a poly(A) tail, and an additional sequence at the end of the poly(A) tail; and (b) a DNA oligo complementarily binding to a portion of the poly(A) tail and an additional sequence at the end of the poly(A) tail, and having a hydrophobic residue introduced therein.

[0033] The mRNA is transcribed from a DNA molecule and essentially includes a coding region encoding a protein to be synthesized, and may include an untranslated region (UTR). The mRNA may include a 5' Cap, and may additionally include a 3' untranslated region (UTR) downstream of the coding region for mRNA expression of the target gene, and may additionally include a 5' untranslated region (UTR) upstream of the coding region for mRNA expression of the target gene.

[0034] The above "5' Cap" is a modified structure that appears to be the 5' end of cytoplasmic mRNA of eukaryotes, and may be involved in the stability of mature mRNA and translation initiation.

[0035] The above "UTR (untranslated region)" refers to the untranslated region of mRNA, generally referring to both ends of the coding region. Specifically, the 5' end is referred to as the 5' UTR, and the 3' end as the 3' UTR. UTRs are known to influence mRNA stabilization and subcellular localization, as well as translation initiation, elongation, and termination, through their interactions with RNA-binding proteins. Depending on specific motifs within the UTR, it can either increase or decrease mRNA turnover.

[0036] In the present invention, the "poly(A) tail", also called polyadenylic acid or polyadenylic acid fragment, refers to a continuous sequence of adenylic acid that is universally present at the 3' end of eukaryotic mRNA. Its length is approximately 10 to 500 nucleotides (nt), and can be variously adjusted depending on the allowable size of the expression vector backbone. Poly(A) can be located downstream of the 3' untranslated region (UTR) and is known to be involved in mRNA stabilization, translation, and transport from the nucleus to the cytoplasm.

[0037] In the present invention, the poly(A) tail may have a length of 10 nt to 500 nt, preferably 10 nt to 200 nt, but is not limited thereto.

[0038] In the present invention, the additional RNA sequence at the end of the poly(A) tail may be any polyribonucleotide sequence or random RNA sequence linked to the 3' end of the poly(A) tail of the mRNA, and may be applied without limitation as long as it is a sequence that increases the protein expression rate of the mRNA without interfering with the UTR or ORF sequence. The additional RNA sequence may be an RNA sequence of any length, and may be 5 to 100 nt, but is not limited thereto.

[0039] In the present invention, a portion of the poly(A) tail sequence may be 2 to 10 nt.

[0040] In the present invention, the DNA oligo comprises a poly(T) tail and complementarily binds to a portion of the poly(A) tail and an additional sequence at the end of the poly(A) tail.

[0041] In the present invention, "complementary binding" may include a sequence consisting of 60 to 100% complementary sequences, preferably 80 to 100% complementary sequences, more preferably 90 to 100% complementary sequences, and even more preferably 95 to 100% complementary sequences, in addition to a 100% complementary sequence, as long as they can maintain the characteristic of forming complementary binding with each other.

[0042] In the present invention, the introduction of the hydrophobic residues can be accomplished through modification of the DNA oligo, and the modification can be accomplished through, but is not limited to, alkylation, cholesterol, cholesterol derivatives, phospholipids, glycolipids, glycerol esters, steroids, ceramides, isoprene derivatives, adamantane, farnesol, aliphatic groups, or polyaromatic compounds. The hydrophobic residues can each be independently introduced into the oligonucleotide through a simple covalent bond or a linker-mediated covalent bond. In addition, the modification can be accomplished at the 5' position, the 3' position, or both, and can be accomplished within them.

[0043] In the present invention, the nucleic acid structure forms a hybrid structure in which a DNA oligo sequence complementary to the poly(A) tail of mRNA or an additional RNA sequence at the end of the poly(A) tail is combined to form a double strand. The hybrid structure may be characterized by comprising 5 to 150 bp, but is not limited thereto.

[0044]

[0045] Additionally, the present invention provides a composition comprising the nucleic acid structure and nanoparticles.

[0046] Additionally, the present invention provides a vaccine composition comprising the nucleic acid structure.

[0047] The mRNA construct may be delivered via a liposome, a lipid nano-particle (LNP), a polymer nanoparticle, or various nanoparticles. The liposome or LNP may include a cationic lipid, a non-cationic lipid, or a neutral lipid, and may additionally include, but is not limited to, other lipids such as PEG (polyethylene glycol) or cholesterol.

[0048] In addition to the above components, the composition of the present invention may further include a lubricant, a wetting agent, a sweetener, a flavoring agent, an emulsifier, a suspending agent, a preservative, etc. Solid preparations for oral administration include tablets, pills, powders, granules, capsules, etc., and these solid preparations are prepared by mixing the ceftezol with at least one excipient, such as starch, calcium carbonate, sucrose or lactose, gelatin, etc. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral administration include suspensions, oral solutions, emulsions, syrups, etc., and in addition to commonly used simple diluents such as water and liquid paraffin, various excipients such as wetting agents, sweeteners, fragrances, preservatives, etc. may be included. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solutions, suspensions, emulsions, lyophilized preparations, and suppositories. Non-aqueous solutions and suspensions can include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. Suppository bases include witepsol, macrogol, Tween 61, cacao butter, laurin, and glycerogelatin.

[0049] The content and administration method of the active ingredients, etc. included in the composition of the present invention can be determined by a person skilled in the art based on the symptoms and severity of the disease of a typical patient. Furthermore, the composition can be formulated in various forms, such as powders, tablets, capsules, liquids, injections, ointments, and syrups, and can also be provided in unit-dose or multi-dose containers, such as sealed ampoules and bottles.

[0050] The composition of the present invention can be administered orally or parenterally. Routes of administration for the composition of the present invention include, but are not limited to, intrabronchial, oral, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intracardiac, transdermal, subcutaneous, intraperitoneal, enteral, sublingual, or topical administration.

[0051] The dosage of the composition according to the present invention varies depending on the patient's weight, age, sex, health condition, diet, administration time, method, excretion rate, or disease severity, and can be easily determined by a person skilled in the art. However, for desirable effects, it is recommended to administer 0.0001 to 100 mg / kg per day, preferably 0.001 to 100 mg / kg. Administration may be administered once a day or divided into several doses. The above dosage does not limit the scope of the present invention in any way. In addition, the composition of the present invention can be formulated into a suitable dosage form using known techniques for clinical administration.

[0052] In addition, the present invention provides a method for producing a nucleic acid structure, comprising: 1) a step of synthesizing mRNA by performing in vitro transcription using a vector containing mRNA including a coding region for mRNA expression of a target gene, a poly(A) tail, and an additional sequence at the end of the poly(A) tail as a template; 2) a step of producing a DNA oligo complementarily binding to a portion of the poly(A) tail sequence and an additional sequence at the end of the poly(A) tail, and introducing a hydrophobic residue; and 3) a step of annealing the mRNA of step 1) and the DNA oligo of step 2).

[0053] In the above step 1), a promoter sequence (T7, SP7, etc.) can be linked to the 5' end of the sequence to produce mRNA of the target gene by an in vitro transcription method in order to express the mRNA of the desired target gene in vitro.

[0054] In the above step 3), the conditions for annealing are sequence-dependent and can be performed under various conditions depending on environmental variables.

[0055] Hereinafter, the present invention will be described in more detail through examples and experimental examples.

[0056] These examples and experimental examples are intended solely to illustrate the present invention, and it will be apparent to those skilled in the art that the scope of the present invention is not to be construed as being limited by these examples.

[0057] <Example 1> Preparation of DNA template for preparation of nucleic acid structure

[0058] A DNA template was prepared by performing PCR (Polymerase chain reaction), and the results were confirmed through electrophoresis.

[0059] Specifically, PCR was performed using a DNA vector (Luciferase-pcDNA3) containing a T7 promoter, 5' UTR, an open reading frame (ORF) encoding the Luciferase protein, and a 3' UTR as a template. The forward primer (FP) was used with the same 18 nt base sequence existing in the front part of the T7 promoter in the template strand of the DNA vector (Luciferase-pcDNA3), and the reverse primer (RP) was used with the 18 nt base sequence complementary to the end of the 3' UTR in the template strand of the DNA vector (Luciferase-pcDNA3) plus 120 nt of a poly(T) tail to introduce a poly(A) tail during IVT mRNA synthesis.

[0060] The sequence of each primer is shown in [Table 1] below.

[0061]

[0062] As a result of performing PCR using the above forward and reverse primers with the DNA vector (Luciferase-pcDNA3) as a template, a DNA template containing a T7 promoter, 5'UTR, open reading frame (ORF), 3'UTR, and a 120 bp poly(dA:dT) tail was synthesized. Since this DNA template encodes the Luciferase protein, it was named Luc (Fig. 1).

[0063] The reverse primer used to introduce the hybrid construct later was designed to have an 18 nt sequence complementary to the end of the 3' UTR of the template strand of the DNA vector (Luciferase-pcDNA3) and a 120 bp poly(dA:dT) tail plus an additional 21 bp or 60 bp of sequence. The DNA template synthesized using the reverse primer with the additional sequence was named Luc21 or Luc60 (Fig. 1).

[0064] The IVT (In VitroTranscription) mRNAs synthesized using DNA templates Luc, Luc21, and Luc60 were named mLuc, mLuc21, and mLuc60, respectively.

[0065] <Example 2> Production of a nucleic acid structure bound to a DNA oligo with a hydrophobic residue (1)

[0066] An annealing process was performed to introduce a DNA oligo complementary to the additional sequence at the 3' end of mLuc21, forming an RNA / DNA hybrid structure. Cholesterol is a lipid present in the body and is suitable as a hydrophobic residue due to its low risk of side effects.

[0067] Specifically, 5'Chol-DNA Oligo (5'-Cholesterol-DNA-3') with cholesterol attached to the 5' end in addition to 27 nt of DNA Oligo (DNA of the sequence complementary to the 21 nt of additional sequence + 6 dT) that complementarily binds to 6 poly (A) tails that are part of the poly (A) tail following 21 nt of additional sequence at the poly (A) tail end of mLuc21, and 5',3'Chol-DNA Oligo (5'-Cholesterol-DNA-Cholesterol-3') with cholesterol attached to both 5' and 3' ends in addition to the 27 nt of DNA Oligo were annealed to mLuc21 at a molar ratio of 1:0.5 (mRNA: Chol-DNA Oligo), thereby producing two mRNAs that introduced a 27 bp RNA / DNA hybrid double strand and cholesterol to the 3' end of mLuc21. They were manufactured and named mLuc21 / 5'Chol and mLuc21 / 5',3'Chol, respectively. Electrophoresis of the nucleic acid structures was performed using a 15% PAGE gel.

[0068] As a result, as shown in Fig. 2a, when the bands of mLuc21 / 5'Chol and mLuc21 / 5',3'Chol after annealing were compared with the bands of each Chol-DNA Oligo (5'Chol-DNA Oligo and 5',3'Chol-DNA Oligo) before annealing, it was confirmed that the Chol-DNA Oligo band that was visible before annealing disappeared in the bands after each annealing. This confirmed that the Chol-DNA Oligo was successfully introduced into mLuc21. Consequently, by annealing each Chol-DNA Oligo to mRNA, an RNA / DNA-Chol hybrid structure was formed, successfully introducing hydrophobicity to the mRNA. Fig. 2b shows the structure of mRNA in which each of the two Chol-DNA Oligos was annealed.

[0069] The sequences of the two Chol-DNA Oligos are shown in [Table 2].

[0070]

[0071] <Example 3> Production of a nucleic acid structure conjugated with a DNA oligo containing a hydrophobic residue (2)

[0072] <3-1> Preparation of alkyl PS (phosphorothioate)-DNA Oligo through Alkylation

[0073] Three types of alkyl PS-DNA Oligo were prepared by alkylating PS-65 DNA Oligo (PS-DNA with a sequence complementary to the 60 nt additional sequence at the end of the poly(A) tail of mLuc60 + 5 PS-T) with phosphorothioate linkages complementary to the 5 poly(A) tails that are part of the poly(A) tail, followed by 8 or 20 dT, which is a DNA oligo of phosphodiester linkages, PS-65A DNA Oligo or PS-65B DNA Oligo. The PS-DNA Oligos before and after alkylation were electrophoresed using a 15% TBE-Urea (8 M) gel.

[0074] As a result, as shown in Fig. 3, the band of the alkyl PS-DNA oligo became lighter compared to the non-alkylated PS-DNA oligo, and it was confirmed that most of the alkyl PS-DNA oligo was caught in the well due to the hydrophobic action of the alkyl group causing the oligos to aggregate with each other. This confirmed that the PS-DNA oligo was alkylated successfully.

[0075] The alkylation mechanism of PS-DNA Oligo is shown in Fig. 4, and the sequences of three PS-DNA Oligos are shown in [Table 3] (*: Phosphorothioate linkage).

[0076]

[0077] <3-2> Preparation of nucleic acid structures introducing alkyl PS-DNA oligos

[0078] Alkyl PS-65 DNA Oligo was annealed to mLuc60 at a molar ratio of 1:1 (mRNA: alkyl PS-DNA Oligo), and alkyl PS-65A DNA Oligo and alkyl PS-65B DNA Oligo were annealed to mLuc60 at a molar ratio of 1:0.5 to produce three mRNAs introducing RNA / DNA hybrid structures to the 3' end of mLuc60, which were designated as mLuc60 / alkyl PS-65, mLuc60 / alkyl PS-65A, and mLuc60 / alkyl PS-65B, respectively. The three mRNAs after annealing and the three PS-DNA Oligos before annealing (PS-65 DNA Oligo, PS-65A DNA Oligo, PS-65B DNA Oligo) were electrophoresed using a 15% PAGE gel.

[0079] As a result, as shown in Fig. 5, when the bands of mLuc60 / alkyl PS-65, mLuc60 / alkyl PS-65A, and mLuc60 / alkyl PS-65B after annealing were compared with the bands of each PS-DNA Oligo (PS-65 DNA Oligo, PS-65A DNA Oligo, PS-65B DNA Oligo) before annealing, it was confirmed that the PS-DNA Oligo bands that were visible before annealing disappeared in the bands after each annealing. This confirmed that the PS-DNA Oligo was successfully introduced into mLuc60. Consequently, by annealing each of the three alkyl PS-DNA Oligos to mRNA, an RNA / alkyl PS-DNA hybrid structure was formed, successfully introducing hydrophobicity into the mRNA.

[0080] <Experimental Example 1> Optimization of the Preparation and Formulation of Lipid Nanoparticles for mRNA Delivery

[0081] The four lipids that make up the lipid nanoparticles (LNPs) are Moderna ® The ionizable lipids are SM-102 (ionizable lipid), Cholesterol, DSPC (helper lipid), and DMG-PEG2K (PEG-lipid), and each lipid is composed of a molar ratio of 50:10:38.5:1.5. The NP ratio, which is the standard for setting the ratio of mRNA and lipids, is the ratio of N (nitrogen) of the ionizable lipid among the four lipids and P (phosphorus) of the mRNA, and the amount of remaining lipid is determined by the molar ratio of the lipids mentioned above (50:10:38.5:1.5) according to the amount of ionizable lipid determined by the NP ratio. Moderna ® The NP ratio of the mRNA vaccine formulation is 5.6 (designated L1).

[0082] Moderna ®When comparing the amount of lipid to mRNA in the formulation, more than 22 times the amount of lipid was delivered into the body along with the mRNA (Figure 6, Table 4). Because the delivery of such a large amount of lipid into the body can lead to various side effects, it was necessary to reduce the amount of lipid to mRNA. Therefore, we designed three formulations with a reduced NP ratio of 3.6 (designated L2), 2.25 (designated L3), and 3.15 (designated L4).

[0083]

[0084] Moderna in Figure 6 ® mRNA vaccine formulations and Moderna ® The total lipid content of formulations with reduced amounts of lipid relative to mRNA is shown. The ratio shown in blue on the left side of the first bar graph is the lipid ratio, which is the same for L1 to L4, and the ratio shown in red on the right side of each bar graph is the NP ratio of each formulation.

[0085] If we successfully form formulations (L2, L3, L4) with reduced amounts of lipids compared to mRNA, the amount of lipids used will be Moderna ® It can be expected that the effect of reducing the amount of lipid in the formulation to less than 50% can be achieved.

[0086] To improve the structural stability and expression efficiency of mRNA by introducing hydrophobicity into mRNA while compensating for the reduced lipid content and increasing the capture efficiency of mRNA within the drug delivery system,

[0087] Figure 7a shows the expected particle-encapsulation form when mRNA without introducing hydrophobicity (mRNA) is encapsulated in lipid nanoparticles, Figure 7b shows the expected particle-encapsulation form when cholesterol is introduced (mRNA / Chol), and Figure 7c shows the expected particle-encapsulation form when alkyl groups are introduced (mRNA / alkyl PS) are encapsulated in lipid nanoparticles.

[0088] Due to the hydrophobic interaction between the cholesterol in mRNA / Chol and other lipids, it was expected that mRNA / Chol would be better captured and encapsulated with a relatively high encapsulation rate than mRNA without cholesterol. In addition, it was expected that lipid nanoparticles encapsulating mRNA without (mRNA) and with (mRNA / alkyl PS) alkyl groups would also be better captured and encapsulated with a relatively high encapsulation rate than mRNA without alkyl groups due to the hydrophobic interaction of the alkyl groups.

[0089] <Experimental Example 2> Confirmation of physicochemical properties of lipid nanoparticles according to mRNA structure and formulation (1)

[0090] Experiments were conducted to determine the physicochemical properties of each lipid nanoparticle encapsulating single-stranded mRNA and mRNA with an RNA / DNA-Chol hybrid structure at the 3' end of the mRNA.

[0091] Specifically, the ratio between mRNA and lipid during encapsulation was set to three different ratios (L1, L2, L3), which were two ratios that reduced the amount of lipid relative to mRNA in addition to the existing ratio. The lipids used for encapsulation were SM-102, Cholesterol, DSPC, and DMG-PEG2K, and the molar ratio of each lipid was 50:10:38.5:1.5, and the volumes of the mRNA solution and the lipid solution were mixed in a ratio of 3:1.

[0092] Using a microfluidic device, Precision Nanosystems' NanoAssemblr® Ignite™, we encapsulated two types of mRNAs: single-stranded mRNA and mRNA with an RNA / DNA-Chol hybrid structure at the 3' end, into lipid nanoparticles. The physicochemical properties (Encapsulation Efficiency%, Diameter Size, PDI) of each lipid nanoparticle encapsulating single-stranded mRNA and mRNA with an RNA / DNA-Chol hybrid structure at the 3' end were determined (Table 5).

[0093]

[0094] As a result, as shown in Fig. 8 and Table 5, when comparing the lipid nanoparticles encapsulated by reducing the amount of lipid relative to mRNA, it was difficult to find a large difference in the encapsulation rate between mRNA with cholesterol introduced (mLuc21 / 5'Chol or mLuc21 / 5'3'chol) and without cholesterol introduced (mLuc) when L2 (NP 3.6), but when L3 (NP 2.25), it was confirmed that the encapsulation rate of mRNA with cholesterol introduced at the 3' end of mRNA (mLuc21 / 5'Chol or mLuc21 / 5'3'chol) was higher than that of single-stranded mRNA without cholesterol introduced (mLuc). Consequently, when cholesterol was introduced into mRNA to increase hydrophobicity, it was confirmed that mRNA was better encapsulated in lipid nanoparticles due to the hydrophobic interaction between the cholesterol bound to mRNA and lipid.

[0095] When comparing the sizes of the lipid nanoparticles of the control group, L1, with those of L2 or L3, no significant differences were observed. Furthermore, the PDI of all lipid nanoparticles was below 0.2, confirming that the particle sizes were consistent across the board. Consequently, we confirmed that lipid nanoparticles were formed even under conditions where the amount of lipid relative to mRNA was reduced for L2 and L3.

[0096] <Experimental Example 3> Confirmation of the physicochemical properties of lipid nanoparticles according to mRNA structure and formulation (2)

[0097] Experiments were conducted to determine the physicochemical properties of each lipid nanoparticle encapsulating single-stranded mRNA and mRNA with an RNA / alkyl PS-DNA hybrid structure at the 3' end of the mRNA.

[0098] Specifically, the ratio between mRNA and lipid during encapsulation was set to the original ratio and a ratio with a reduced amount of lipid compared to mRNA (L1, L2, L4). The lipids used for encapsulation were SM-102, DSPC, Cholesterol, and DMG-PEG2K, and the molar ratio of each lipid was 50:10:38.5:1.5, and the volumes of the mRNA solution and the lipid solution were mixed at a ratio of 3:1.

[0099] Using a microfluidic device, Precision Nanosystems' NanoAssemblr® Ignite™, we encapsulated single-stranded mRNA and three mRNAs with RNA / alkyl PS-DNA hybrid structures at the 3' end into lipid nanoparticles. The physicochemical properties (Encapsulation Efficiency%, Diameter Size, PDI) of each lipid nanoparticle encapsulating single-stranded mRNA and mRNA with RNA / alkyl PS-DNA hybrid structures at the 3' end were determined (Table 6).

[0100]

[0101] As a result, as shown in Fig. 9 and Table 6, when comparing the lipid nanoparticles encapsulated by reducing the amount of lipid relative to mRNA, it was confirmed that the encapsulation rate of mRNA with an alkyl group introduced at the 3' end of mRNA (mLuc60 / alkyl PS-65, mLuc60 / alkyl PS-65A, mLuc60 / alkyl PS-65B) was higher than that of single-stranded mRNA (mLuc) without an alkyl group introduced when both L2 (NP 3.6) and L4 (NP 3.15) were used. Consequently, it was confirmed that when an alkyl group was introduced to mRNA to increase hydrophobicity, the mRNA was better encapsulated in the lipid nanoparticles due to the hydrophobic interaction between the alkyl groups bound to the mRNA.

[0102] In addition, when comparing the sizes of the lipid nanoparticles of the control group L1 with those of L2 or L4, no significant differences were observed. The PDI of all lipid nanoparticles was less than 0.2, confirming that the sizes of the particles were consistent throughout.

[0103] <Experimental Example 4> Confirmation of protein expression levels according to mRNA structure and formulation (1)

[0104] An experiment was conducted to determine the level of protein expression by transfecting cells with each lipid nanoparticle encapsulating mRNA having a single-stranded mRNA and an RNA / DNA-Chol hybrid structure in which a DNA oligo with cholesterol attached to the 3' end of the mRNA was introduced.

[0105] Specifically, HEK-293T cells (Human embryonic kidney-293T) and HeLa cells were seeded at 2x10 in 96-well plates, respectively. 4 cells / well, 1x10 4After seeding cells / well and culturing for more than 18 hours, lipid nanoparticles encapsulating single-stranded mRNA (mLuc) encoding Firefly Luciferase and lipid nanoparticles encapsulating mRNA with cholesterol introduced at the 3' end (mLuc21 / 5'Chol or mLuc21 / 5'3'chol) were transfected into cells at a dose of 70 ng / well, and after 18 hours, the expression of Firefly Luciferase protein was measured using a Luciferase assay system (Bright-Glo™ Luciferase Assay System, Promega, USA). The higher the protein expression efficiency, the higher the Luciferase Activity (RLU, Relative Light Unit) value. Moderna was used as a positive control in the measurement of protein expression efficiency of mRNA-encapsulated lipid nanoparticles. ® Lipid nanoparticles encapsulating mLuc without cholesterol were used as a formulation (L1 mLuc LNP).

[0106] As a result, in the case of lipid nanoparticles encapsulating mRNA (mLuc21 / 5'Chol or mLuc21 / 5'3'chol) with cholesterol introduced at the 3' end of mRNA, as shown in Fig. 10a and Fig. 10b, the positive control (Moderna ® Despite a 50% reduction in lipid content compared to the positive control (Moderna) the protein expression efficiency was higher ® It was confirmed that the protein expression efficiency was maintained similarly to that of the formulation.

[0107] <Experimental Example 5> Confirmation of protein expression levels according to mRNA structure and formulation (2)

[0108] An experiment was conducted to encapsulate single-stranded mRNA and mRNA with an RNA / alkyl PS-DNA hybrid structure with an alkyl group introduced at the 3' end of the mRNA into lipid nanoparticles (LNPs), and then transduce each lipid nanoparticle into cells to determine the level of protein expression.

[0109] Specifically, HepG2 cells (Hepatocellular carcinoma, HCC) were seeded at 2x10 in a 96-well plate. 4 After seeding cells / well and culturing for more than 18 hours, lipid nanoparticles encapsulating single-stranded mRNA (mLuc) encoding Firefly Luciferase and lipid nanoparticles encapsulating mRNA with an alkyl group introduced at the 3' end of mRNA (mLuc60 / alkyl PS-65A, mLuc60 / alkyl PS-65B) were transfected into cells at a dose of 70 ng / well, and after 18 hours, the expression of Firefly Luciferase protein was measured using a Luciferase assay system (Bright-Glo™ Luciferase Assay System, Promega, USA). Moderna was used as a positive control for measuring protein expression efficiency of mRNA-encapsulated lipid nanoparticles. ® Lipid nanoparticles encapsulating mLuc without introducing alkyl groups into the formulation were used (L1 mLuc LNP).

[0110] As a result, as shown in Fig. 11, in the case of lipid nanoparticles encapsulating mRNA with alkyl groups introduced at L2 (mLuc60 / alkyl PS-65A, mLuc60 / alkyl PS-65B) and mLuc60 / alkyl PS-65A at L4, the protein expression efficiency was higher than that of the positive control group (Moderna ®Although lower than that of the positive control (Moderna), in the case of lipid nanoparticles encapsulating mLuc60 / alkyl PS-65B at L4, the protein expression efficiency was higher than that of the positive control (Moderna ® Despite a 40% reduction in lipid content compared to the positive control (Moderna) the protein expression efficiency was higher ® It was confirmed that the protein expression efficiency was maintained similarly to that of the formulation.

Claims

1. (a) mRNA comprising a coding region for mRNA expression of a target gene, a poly(A) tail, and additional sequences at the end of the poly(A) tail; and (b) a DNA oligo complementarily binding to a portion of the poly(A) tail and an additional sequence at the end of the poly(A) tail, and having a hydrophobic residue introduced therein; A nucleic acid structure comprising:

2. In paragraph 1, A nucleic acid structure characterized in that the introduction of the hydrophobic residue is accomplished through modification of the DNA oligo, wherein the modification is accomplished through alkylation, cholesterol, cholesterol derivatives, phospholipids, glycolipids, glycerol esters, steroids, ceramides, isoprene derivatives, adamantane, farnesol, aliphatic groups or polyaromatic compounds.

3. In paragraph 1, A nucleic acid structure, characterized in that the poly(A) tail has a length of 10 nt to 500 nt.

4. In paragraph 1, A nucleic acid structure characterized in that the additional sequence at the end of the poly(A) tail is 5 nt to 100 nt.

5. In paragraph 1, A nucleic acid structure, characterized in that a portion of the poly(A) tail sequence is 2 nt to 10 nt.

6. In paragraph 1, A nucleic acid structure, characterized in that the above DNA oligo comprises a poly(T) tail.

7. In paragraph 1, A nucleic acid structure characterized in that the mRNA structure additionally includes a 3' untranslated region (UTR) downstream of a coding region for mRNA expression of a target gene.

8. In paragraph 1, A nucleic acid structure characterized in that the mRNA structure additionally includes a 5' untranslated region (UTR) upstream of a coding region for mRNA expression of a target gene.

9. A composition comprising a nucleic acid structure and a lipid nanoparticle according to any one of claims 1 to 8.

10. A vaccine composition comprising a nucleic acid structure of any one of claims 1 to 8. 11.1) A step of synthesizing mRNA by performing in vitro transcription using a vector containing mRNA including a coding region for mRNA expression of a target gene, a poly(A) tail, and an additional sequence at the end of the poly(A) tail as a template; 2) a step of producing a DNA oligo complementarily binding to a portion of the poly(A) tail sequence and an additional sequence at the end of the poly(A) tail, and introducing a hydrophobic residue; and 3) Step of annealing the mRNA of step 1) and the DNA oligo of step 2); A method for producing a nucleic acid structure, comprising:

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